High-resolution analysis of cytosine methylation in ancient DNA

Abstract

Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution.

Figure 1. Methylation patterns of repetitive genomic elements in modern and ancient bovids.

Maps of CpG methylation (white squares, unmethylated; black squares, methylated) composed of 20 individual clones (horizontal lines) were generated from Bos taurus, mummified Bos taurus and ancient Bison priscus targeting the multicopy retrotransposons as indicated above the maps. Differences in CpG placement within the retrotransposon maps are due to amplification of closely related but not identical elements from distinct locations within the genome. Retrotransposon consensus sequences were obtained from Repbase .

Figure 2. Methylation patterns of single copy imprinted genes in modern and ancient bovids.

Maps of CpG methylation (white squares, unmethylated; black squares, methylated) composed of 20 individual clones (horizontal lines) were generated from Bos taurus, mummified Bos taurus and ancient Bison priscus targeting the single-copy imprinted genes PEG3 (GenBank AY427787) and NESP55 (GenBank U77614). Clone sequences are shown in Fig. S2, S3, S4.

Figure 3. The potential effect of spontaneous post-mortem deamination of cytosine residues on the resolution of methylation patterns.

In fresh samples (left), methylated cytosine is resistant to bisulphite conversion and can be resolved after PCR and sequencing. Post-mortem deamination (DNA damage) is highlighted by arrowheads in the ancient sample (right). A proportion of unmethylated cytosine in ancient templates is sometimes converted to uracil and appears as thymine following PCR amplification ; this would not affect the resolution of methylation. However, post-mortem deamination of methylated cytosine to thymine would prevent the detection of methylation via bisulphite sequencing (circles).